Antony Citterio-Quentin

Determination of inosine triphosphate pyrophosphatase phenotype in human red blood cells using HPLC.

Background: Thiopurine drugs, widely used in cancer chemotherapy, inflammatory bowel disease, and autoimmune hepatitis, are responsible for common adverse events. Only some of these may be explained by genetic polymorphism of thiopurine S-methyltransferase. Recent articles have reported that inosine triphosphate pyrophosphatase (ITPase) deficiency was associated with adverse drug reactions toward thiopurine drug therapy. Here, we report a weak anion exchange high-performance liquid chromatography method to determine ITPase activity in red blood cells and to investigate the relationship with the occurrence of adverse events during azathioprine therapy. Methods: ITPase activity was assessed by the enzymatic conversion of inosine triphosphate (ITP) to inosine monophosphate (IMP). The reaction was stopped by heating for 3 minutes at 120°C. IMP, inosine diphosphate, and ITP were analyzed on a Hypersil APS-2 column, a weak anion exchange phase that exhibits both ionic and hydrophobic properties. Results: The chromatographic method reported allows the analysis of IMP, inosine diphosphate, and ITP in a single run in <12.5 minutes. The method was linear in the range 5–1500 &mgr;mole/L of IMP. Intraassay and interassay precisions were <5% for red blood cell lysates supplemented with 50, 500, and 1000 &mgr;mole/L IMP. Km and Vmax evaluated by Lineweaver–Burk plot were 677.4 &mgr;mole/L and 19.6 &mgr;mole·L−1·min−1, respectively. The frequency distribution of ITPase from 73 patients was investigated. Conclusions: The method described is useful to determine the ITPase phenotype from patients on thiopurine therapy and to investigate the potential relation between ITPase deficiency and the occurrence of adverse events.

Determination of inosine triphosphate pyrophosphatase in red blood cells using HPLC: an improved method.

To the Editors:The thiopurine drugs, azathioprine (AZA), and 6-mercaptopurine (6-MP) are well-established immunosuppressive agents that are used successfully in the treatment of a range of diseases. In particular, azathioprine therapy has an important role in the induction and maintenance of remissi

ITPA Activity in Adults and Children Treated With or Without Azathioprine: Relationship Between TPMT Activity, Thiopurine Metabolites, and Co-medications

Background: The implication of inosine triphosphate pyrophosphatase (ITPA) on thiopurine drug response variability has been investigated but little data are available on its role on thiopurine metabolites. The ability of ITPA to modify the thiopurine metabolite levels is currently used to optimize azathioprine (AZA) therapy in relation to thiopurine S-methyltransferase (TPMT) activity, the aim of this study is to investigate ITPA phenotype in a large population and to evaluate the relation between ITPA and TPMT activities and thiopurine metabolites. Methods: ITPA activity was determined in 183 adults and 138 children with or without AZA therapy. 6-thioguanine nucleotides (6-TGN), 6-methylmercaptopurine nucleotides (6-MeMPN) levels, and ITPA as well as TPMT activities were measured in red blood cells. Using the Gaussian mixture model, distribution of ITPA activity was evaluated. Intraindividual variability and influence of age, sex, AZA treatment and associated co-medications on ITPA activity were also assessed. Results: This retrospective study shows a quadrimodal distribution in ITPA activity. No influence of age, sex, AZA therapy, and co-medications was found. In adults, ITPA activity was not significantly associated with 6-TGN or 6-MeMPN concentrations, whereas a weak negative correlation was observed with 6-MeMPN levels in pediatric populations (rs = −0.261; P = 0.024). A weak positive correlation was observed between ITPA and TPMT activities in children (rs = 0.289; P = 0.001). Conclusions: ITPA activity was poorly influenced by nongenetic parameters and has no influence on 6-TGN and 6-MeMPN concentrations in adults and only a weak correlation with 6-MeMPN and TPMT activity in children. These results demonstrate that ITPA is not a rate-limiting enzyme in the formation of 6-TGN but suggest that a decrease in ITPA activity in children may be a risk factor for accumulation of 6-MeMPN in cells.

LSD Screening in Urine Performed by CEDIA® LSD Assay: Positive Interference with Sertraline

To the Editor: Although more specific than the enzyme multiplied immunoassay technique (EMIT) (1), cloned enzyme donor immunoassay (CEDIA)(Microgenics Corp., Fremont, CA) lysergic acid diethylamide (LSD) screening in urine is subject to various cross-reactions, particularly with ambroxol (2) and fentanyl (3). Following a clinical observation, we carried out several tests to confirm a cross-reaction with sertraline [one of the most prescribed antidepressants, particularly in the United States (4)], which, to our knowledge, has not been described in the literature. A 16-year-old boy suffering from abdominal pain, diarrhea, vomiting, headache and tremors presented to Lyon hospital’s pediatric emergency department. He claimed not to have taken drugs. He had been treated for social phobia for three months with a combination of sertraline (Zoloft 50 mg) and hydroxyzine (Atarax 25 mg). The clinical examination showed a serotonin syndrome (sinus tachycardia, hypertension: 168/70 mmHg, excessive sweating, extra-cellular dehydration and hyperreflexia in the lower limbs with myoclonic jerks) associated with visual hallucinations. The toxicological screenings confirmed he had taken Zoloft. After a broadspectrum screening in serum by high-performance liquid chromatography–diode-array detection (HPLC–DAD) that revealed a 0.27 mg/L supra-therapeutic serum concentration of sertraline [therapeutic concentrations: 0.05–0.25 mg/L (TIAFT: The International Association of Forensic Toxicologists)], we measured sertraline (as a parent compound) in urine by HPLC–DAD/mass spectrometry (MS) (m/z 308) and found a 1.91 mg/L urinary concentration. Moreover, the LSD screening in urine using the CEDIA assay was positive. In a new interview with the medical team, the boy confirmed that he had taken four tablets of sertraline 12 hours earlier, but the positive LSD result remained unexplained. His family assured them he was unable to obtain LSD because he rarely left home. Despite the clinical compatibility, the LSD screening in urine was secondarily verified by HPLC–DAD/MS [LSD (m/z 324) and nor LSD (m/z 310) limits of quantification 1⁄4 1.0 mg/L]. The result was negative, suggesting possible interference, so we decided to test. We fortified urine samples from a control subject who had not taken LSD with sertraline (concentration range of 0–2 mg/ L). On each of the fortified samples, we performed an LSD screening in urine with the CEDIA assay using two different batches of reagent and confirmed the result with HPLC–DAD/ MS. We also considered the case of a second patient with a sertraline overdose (serum concentration: 1.43 mg/L, urinary concentration: 1.78 mg/L) and two other cases of patients receiving long-term sertraline treatment (serum concentrations: 0.120 and 0.145 mg/L, urinary concentrations: 0.530 and 1.20 mg/L). On the urine samples from the latter cases, we performed LSD screening with the CEDIA assay and confirmed the results with HPLC–DAD/MS. The results obtained from the urine samples fortified with sertraline showed that the CEDIA LSD screening was positive when concentrations reached 1.5 mg/L (see Table I). We also obtained false-positive LSD screenings with the CEDIA assay in the urine samples from the second case of overdose, as well as in the sertraline-treated patients whose serum concentrations were within the accepted therapeutic range. All of these results were consistent with the observation of the initial case. Thus, the CEDIA assay for LSD screening in urine is not only sensitive to positive interferences related to a slight sertraline overdose, but also in samples from patients receiving longterm sertraline treatment. This interference seems to be attributable to a cross-reaction with sertraline, as shown by our fortified samples, and probably its urinary metabolites. Few data regarding sertraline metabolism in humans are available. However, a study showed that, in animals, less than 0.2% of sertraline is excreted as a parent compound (5). The urinary concentrations of sertraline (parent compound) found in our patients tend to indicate this is not the case in humans. To conclude, a positive LSD CEDIA screening in urine must be confirmed by a specific separation method when a sertraline overdose or long-term sertraline treatment is suspected. In addition, because sertraline is among the most prescribed antidepressants, this interference can be frequently detected. Additional testing is needed to determine whether this interference occurs with other structurally similar compounds.

ITPA Activity in Children Treated by Azathioprine: Relationship to the Occurrence of Adverse Drug Reactions and Inflammatory Response

Azathioprine (AZA), a thiopurine drug, is widely used in the treatment of children with immunological diseases such as inflammatory bowel disease (IBD) and autoimmune hepatitis (AIH); however, interindividual variability in the occurrence of adverse drug reactions (ADRs) and drug response is observed. This study investigated (i) the relationships between inosine triphosphate pyrophosphatase (ITPA) activity, an enzyme involved in thiopurine metabolism, and the occurrence of ADRs in children with immunological disease on AZA therapy, and (ii) the relationship between ITPA activity and the inflammatory activity observed in children with IBD. ITPA and TPMT activities were determined in 106 children with immunological disease on AZA therapy. Markers of hepatotoxicity, myelotoxicity, pancreatitis and inflammation as well as clinical information were retrospectively collected during regular medical visits. No significant association was found between ITPA activity and hepatotoxicity or clinical ADRs such as cutaneous reactions, arthralgia, flulike symptoms and gastrointestinal disorders. Concerning myelotoxicity, a significant relation was observed between ITPA activity and RBC mean corpuscular volume (MCV; p=0.003). This observation may be related to the significant relationship found between high ITPA activity and the increase in γ‐globulin level reflecting inflammation (p=0.005). In our study, ITPA activity was not associated with occurrence of ADRs, but a relationship between high ITPA activity and γ‐globulin, a marker of inflammation, was found in children with IBD. Therefore, measurement of ITPA activity may help to identify children with IBD predisposed to residual inflammation on AZA therapy. Further prospective studies are needed to confirm this result.